Production and Use of Bacterial Histamine

ABSTRACT

A method is provided of selecting specific probiotic lactic acid bacteria producing histamine and the use of such strains for beneficial effects for mammals. The method includes selecting a lactic acid bacterial strain for use in the local production of histamine in a mammal, and further comprises screening bacteria for the presence of an active histidine operon and selecting a strain which has an active histidine operon and is capable of producing histamine. Preferably said strain is selected for its ability to produce histamine at a level of greater than 250 pg/ml. The present invention further provides products comprising the strains obtainable by the selection methods of the invention for use in the local production of histamine in a mammal, in particular for use in the treatment or prophylaxis of inflammatory conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/572,742 filed Jul. 21, 2011.

FIELD OF THE INVENTION

This invention relates to a method of selecting specific probioticlactic acid bacteria producing histamine and the use of such strains todeliver beneficial effects for the host.

BACKGROUND OF THE INVENTION

The Food and Agricultural Organization of the United Nations defineprobiotics as “live microorganisms which when administered in adequateamounts confer a health benefit on the host”. Nowadays, a number ofdifferent bacteria are used as probiotics for example, lactic-acidproducing bacteria such as strains of Lactobacillus and Bifidobacteria.

Lactic-acid producing bacteria are not only used for their beneficialeffect on human or animal health, but they are also widely used in thefood industry for fermentation processes. The effectiveness ofprobiotics is strain-specific, and each strain may contribute to hosthealth through different mechanisms. Probiotics can prevent or inhibitthe proliferation of pathogens, suppress production of virulence factorsby pathogens, or modulate the immune response in a pro-inflammatory oran anti-inflammatory way. Use of different strains of the probioticlactic-acid producing bacteria Lactobacillus reuteri is a promisingtherapy for the amelioration of infantile colic, alleviation of eczema,reduction of episodes of workplace illness, and suppression ofHelicobacter pylori infection. L. reuteri is considered an indigenousorganism of the human gastrointestinal tract and is present for exampleon the mucosa of the gastric corpus, gastric antrum, duodenum, andileum. See, for example, U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253,5,837,238, and 5,849,289.

When L. reuteri cells are grown under anaerobic conditions in thepresence of glycerol, they produce the antimicrobial substance known asreuterin (β-hydroxy propionaldehyde).

The relationship between a host and its microbes is complex, and forsome bacteria, this host: microbe relationship has been developing overmany years of co-evolution. This appears to be especially true forLactobacillus reuteri. Our knowledge of the mutualistic relationshipbetween gut microbes and the human host is in its infancy, but alreadywe are keenly aware that the gut microbiome plays an essential role ingut and immune system development, nutrition, and new links are beingestablished between the gut microbiome and the brain. Dysbiosis, theperturbation of the normal gut microbiome, has been implicated in a widerange of disease processes including those affecting the local gutenvironment, such as Inflammatory Bowel Disease (IBD) and IrritableBowel Syndrome (IBS), and disease processes at sites distant to the gut,such as the metabolic syndrome. Significant therapeutic potential lieswithin the gut microbiome, and research is striving towards a futuregoal of altering the microbial community in order to prevent and/ortreat distinct disease processes.

There is therefore a need to understand such specific interactionsbetween microbes and man related to a specific disease or othersituations influencing the health of the host so that the mostappropriate probiotic strains can be selected and used to counteractsuch developments.

SUMMARY OF THE INVENTION

The invention herein provides a specific method of locally producinghistamine in mammals, especially humans, the local production ofhistamine includes but are not limited to production in the GI tract,genitourinary (GU) tract, oral cavity, in the lungs and airways, on theskin etc, of the mammalian body by selecting certain strains of lacticacid bacteria. The bacteria may be delivered together with certain aminoacids and/or sugars, separately administered or already present at theactive site.

A primary object of the present invention is to select strains that canlocally produce histamine in various locations, including the GI tract,GU tract, oral cavity, in the lungs and airways, on the skin etc, of themammalian body.

It is a further object of the invention to provide products containingsaid strains.

It is a further object of the invention to combine the administration ofbacteria with administration of histidine, or histidine-containing foodsor compositions, to ensure local generation of histamine.

The present invention thus relates to a new method for selecting lacticacid bacterial cells which are useful as probiotics and in therapy. Thisnew method involves the screening and selection for strains of lacticacid bacteria which have an active histidine operon and are capable ofproducing histamine. Surprisingly, the lactic acid bacterial strainsselected by this method are useful as probiotics and in therapy, inparticular in producing anti-inflammatory effects, by way of the localproduction of histamine. These effects of the bacteria are surprising,as discussed elsewhere herein, previously the presence in foodstuffs ofbacteria producing histamine was actively avoided due to the recognizedhealth risk, for example potential toxic effects. Thus, theadministration to a mammal of lactic acid bacteria capable of localproduction of histamine, or indeed the screening and selection of lacticacid bacteria for such capability of local production of histamine basedon the presence of an active histidine operon and an ability to producehistamine is counter-intuitive to this teaching. Indeed, probiotics havenever before been reported to produce histamine.

Thus, at its broadest, the present invention provides a method ofselecting a lactic acid bacterial strain for use in the local productionof histamine in a mammal, wherein said method comprises screeningbacteria for the presence of an active histidine operon and selecting astrain which has an active histidine operon and is capable of producinghistamine.

The histidine operon comprises three genes (the histidine/histamineantiporter, the histidine decarboxylase pyruvoyl type A (HdcA), andhistidine decarboxylase pyruvoyl type B (HdcB)). It is believed that theactivity of each of these genes is important for the present invention.Thus, in the screening methods of the invention, candidate bacteria areassessed for the presence of all three genes and strains positive forall three genes are selected. Any appropriate method can be used for thedetection of the presence of all three genes, for example geneticmethods such as PCR can be used. The production of good levels ofhistamine can also be an indicator of the presence of all three genesand the presence of an active histidine operon. Thus, the selectionmethod of the invention also involves the step of selecting a strainwhich is capable of producing histamine. Strains with high productionlevels of histamine are preferred. Thus, in preferred embodiments astrain is selected for its ability to produce histamine at a level ofgreater than 200, preferably greater than 250 or more preferably greaterthan 300 pg/ml, for example a level of greater than 350, 400, 450, or500 pg/ml. Such values generally refer to values of histamine measuredin the supernatant of strains in culture.

Appropriate methods of measuring levels of histamine production would bewell known to a person skilled in the art. The method of massspectrometry, more specifically triple quadrupole mass spectrometry, isexemplified herein and is preferred. However, equally ELISAs orimmunoassays can be used to evaluate and quantify histamine production.Thus, in some embodiments of the invention, the selection method willinvolve the step of detecting the amount or level of histamine producedby a candidate strain. Because of the downstream uses of the strainswhich are selected by the methods of the invention, afterhistamine-producing strains are selected or isolated, other embodimentswill involve the further steps of culturing or propagating such strains,or possibly storing such strains for future uses.

Such further steps (and indeed the selection steps of the methods of theinvention) will generally need to be carried out in an appropriateculture medium which supports histamine production. Preferred culturemedia will contain an appropriate carbon source which will support theproduction of histamine by said strain. In particularly preferredembodiments, the media will comprise glucose as a carbon source andpreferably will not contain sucrose, or at least will only comprisesucrose at such a level which will not significantly compromisehistamine production by the strain. Histidine or a histidine analog canalso be provided, optionally together with sources of other amino acids.

In preferred embodiments said strain is a strain of Lactobacillusreuteri.

Once an appropriate strain has been selected using the method of thepresent invention it can then be used for the local production ofhistamine in a mammal. Said strains thus also have to be capable oflocal production of histamine in a mammal.

Thus, a further aspect of the present invention provides a productcomprising cells of a lactic acid bacterial strain obtainable by theselection method of the invention, wherein said lactic acid bacterialstrain has an active histidine operon and is capable of producinghistamine, for use in the local production of histamine in a mammal. Aswill be outlined elsewhere herein, preferred uses are in the treatmentand/or prophylaxis of inflammatory conditions, or in the treatmentand/or prophylaxis of conditions or diseases which will benefit fromlocal histamine production. For example, such local production ofhistamine can result in an anti-inflammatory effect.

Alternative embodiments of the invention provide a lactic acid bacterialstrain which is capable of producing histamine for use in the localproduction of histamine in a mammal, wherein said lactic acid bacterialstrain has an active histidine operon. Preferred features of this strainand its uses are described elsewhere herein.

Methods of treatment or methods for the local production of histamine ina mammal, are also provided, said methods comprising the administrationof a product comprising cells of a lactic acid bacterial strainobtainable by the selection method of the invention, or theadministration of a lactic acid bacterial strain wherein said lacticacid bacterial strain has an active histidine operon and is capable ofproducing histamine, to said mammal in an amount effective to enablelocal production of histamine in said mammal. Preferred features of thestrain and its therapeutic uses are described elsewhere herein.

Also provided by the present invention is the use of a productcomprising cells of a lactic acid bacterial strain obtainable by theselection method of the invention, wherein said lactic acid bacterialstrain has an active histidine operon and is capable of producinghistamine, in the manufacture of a composition or medicament for use inthe local production of histamine in a mammal. Alternative embodimentsprovide the use of a lactic acid bacterial strain, wherein said lacticacid bacterial strain has an active histidine operon and is capable ofproducing histamine, in the manufacture of a composition or medicamentfor use in the local production of histamine in a mammal. Preferredfeatures of the strain and its therapeutic uses are described elsewhereherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Quantification of histamine in HILIC-HPLC fractions. Triplequadrupole mass spectrometry was used to quantify histamine present in aselect range of HILIC-HPLC fractions. TNF-inhibitory fractions had thehighest amounts of histamine out of all the fractions examined.

FIG. 2—Purified histamine and histamine from L. reuteri 6475 inhibit TNFproduction via the histamine H₂ receptor. A. Purified histaminesignificantly inhibited TNF production, an effect that is blocked byspecific H₂ receptor antagonists in a dose-dependent manner. Conditionedmedia (or supernatant) containing secreted factors from strain 6475(including histamine) significantly inhibited TNF production, an effectthat is partially blocked by specific H₂ receptor antagonists. N=3, *pvalue <0.05 compared to media control, **p value <0.05 compared tohistamine, p value <0.05 compared to ATCC 6475 conditioned media (CM) B.The cell pellet wash from strain 6475 containing histamine suppressedTNF production, an effect that was partially blocked by specific H₂receptor antagonists. Fraction B3, which contains relatively purehistamine, inhibited TNF production, an effect that was completelyblocked by specific H₂ receptor antagonists. N=3, *p value <0.05compared to media control, **p value <0.05 compared to ATCC 6475 cellpellet wash (CP), ***p value <0.05 compared to fraction B3.

FIG. 3—The histidine operon is important for the TNF-inhibitoryphenotype of L. reuteri 6475. A. The histidine operon consists of threegenes, the histidine/histamine antiporter, hdcA, and hdcB. B. Mutationin any one gene in the histidine operon results in a partial loss of TNFsuppression by L. reuteri 6475. N=9, *p value <0.05 compared to mediacontrol, **p value <0.05 compared to ATCC PTA 6475.

FIG. 4—L. reuteri 6475 significantly reduced weight loss induced by TNBSchallenge, the figure represents data from two independentexperiments, * p<0.05, ** p<0.01, *** p<0.001.

FIG. 5—L. reuteri 6475 significantly diminished macroscopic colon damageinduced by TNBS challenge, the figure represents data from twoindependent experiments, *p<0.05, ***p<0.001.

FIG. 6—L. reuteri 6475 significantly reduced SAA concentration inducedby TNBS challenge, the figure represents data from two independentexperiments, * p<0.05, *** p<0.001.

FIG. 7—hdcA mutant yielded diminished ability to attenuate colitis, thefigure represents data from two independent experiments, * p<0.05, **p<0.01.

FIG. 8—hdcA mutant yielded diminished ability to attenuate colitis, thefigure represents data from two independent experiments, * p<0.01, **p<0.001.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The inventors herein have found that a selected group of lactobacilli,including certain strains of Lactobacillus reuteri locally produceshistamine under specific growth conditions, and that such producedhistamine will benefit the host by for example reducing inflammation,reducing certain cancers etc.

Histamine

Histamine is an organic nitrogen compound involved in severalhealth-associated processes of a mammal, including local immuneresponses as well as regulating physiological function in the gut andacting as a neurotransmitter. As part of an immune response to foreignpathogens, histamine is produced by basophils and mast cells. Histaminecan be derived from the decarboxylation of the amino acid histidine, areaction catalyzed by the enzyme L-histidine decarboxylase.

Bacteria are capable of producing histamine using histidinedecarboxylase enzymes unrelated to those found in eukaryotes. Up to now,such production of histamine by certain bacterial strains has been seenas a health risk rather than a possible benefit for humans. For example,Scombroid poisoning, a form of non-infectious foodborne disease, is dueto histamine production by bacteria in spoiled food, particularly fish.Fermented foods and beverages naturally contain small quantities ofhistamine due to a similar conversion performed by fermenting bacteriaor yeasts. Delivery of certain controlled amounts of histamine fromselected bacteria can, surprisingly, give beneficial effects rather thandetrimental effects as might be expected from the above mentionedstudies.

Histamine receptors are a class of G protein-coupled receptors withhistamine as their endogenous ligand. There are four known histaminereceptors; H₁ receptor (H1R), H₂ receptor (H2R), H₃ receptor (H3R) andH₄ receptor (H4R).

Vannier et al. (Histamine Suppresses Gene Expression and Synthesis ofTumor Necrosis Factor α via Histamine H ₂ Receptors; J Exp Med. 1991July 1; 174(1):281-4) showed that LPS-induced synthesis of TNF-α inperipheral blood mononuclear cells was suppressed by histamine and theyfurther suggest that histamine release from mast cells may limit theextent of inflammatory and immune reactions by suppressing localcytokine synthesis in H₂ receptor-bearing cells.

The anti-inflammatory activity of histamine has previously beendisclosed by Wang et al. (Histamine Antagonizes Tumor Necrosis Factor(TNF) Signaling by Stimulating TNF Receptor Shedding from the CellSurface and Golgi Storage Pool; J. Biol. Chem. 278(24): 21751-21760),showing that histamine causes transient loss of surface TNFR1, increasedTNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi incultured human endothelial cells. Histamine injection into human skinengrafted on immunodeficient mice caused shedding of TNFR1 anddiminished TNF-mediated induction of endothelial adhesion molecules.

Vannier et al. and Wang et al. did not mention anything about usinghistamine-producing bacterial strains as probiotics nor how to selectthe strains based on their histamine-producing abilities in order toassure certain health benefits for the host, such as anti-inflammatoryeffects.

Ceplene, a pharmaceutical-grade form of histamine dihydrochloride, isused for the prevention of relapse in patients diagnosed with acutemyeloid leukemia (AML). Ceplene is administered in conjunction with lowdoses of the immune-activating cytokine interleukin-2 (IL-2) in thepost-remission phase of AML, i.e. when patients have completed theinitial chemotherapy. Studies have shown that Ceplene/IL-2 can induceimmune-mediated killing of leukemic cells. The treatment, subcutaneousinjections, is given in 3-week cycles by the patients at home for 18months. The side effects of Ceplene include transient flush andheadache. It would be advantageous for patients to receive locallyproduced histamine, when needed, instead of subcutaneous injections;this delivery strategy may be achieved by administeringbacterial-derived histamine to the patient using the strains selectedaccording to this invention.

It is previously known that gram-negative bacteria form histamine forexample in raw fish and meat following temperature abuse and thatgram-positive bacteria cause histamine spoilage of fermented foods suchas cheese, sausage, miso, soy sauce, beer and wine. The identificationof histamine-producing bacteria in foods has been difficult.

Also Lactobacillus reuteri has previously been associated with histamineproduction, Casas et al. (Validation of the Probiotic Concept:Lactobacillus reuteri Confers Broad-spectrum Protection against Diseasein Humans and Animals.; 2000, ISSN 0891-060X) reports that two strainsof L. reuteri in the hands of Straub et. al. (Z Lebensm Unters Forsch(1995) 201: 79-82) has been shown to decarboxylate L-histidine to formhistamine and the authors warn against using such strains for thefermentation of food and as probiotics.

Trip et al. (HdcB, a novel enzyme catalyzing maturation ofpyruvoyl-dependent histidine decarboxylase; Molecular Microbiology(2011) 79(4), 861-871) referring to three types of genetic organizationof histidine decarboxylation loci among histamine-producingGram-positive bacteria. The largest group is found in the lactic acidbacteria including L. hilgardii 0006, L. buchneri B301, L. reuteri F275and T. halophilus. Lactobacillus hilgardii 0006 has been shown toproduce histamine, in a study performed by Lucas et al.(Histamine-Producing Pathway Encoded on an Unstable Plasmid inLactobacillus hilgardii 0006; APPLIED AND ENVIRONMENTAL MICROBIOLOGY,March 2005, Vol. 71, No. 3, p. 1417-1424), they further say thathistamine is a contaminant that appears in several products duringgrowth of undesirable bacteria. Lucas et al. have performed a screeningof a collection of wine lactic acid bacteria to identify the genesinvolved in the histamine-producing pathway of a gram-positive bacteriumof wine.

A histamine-producing strain of Lactobacillus buchneri has been isolatedfrom Swiss cheese that had been implicated in an outbreak of histaminepoisoning (Summer et al. Isolation of histamine producing Lactobacillusbuchneri from Swiss cheese implicated in a food poisoning outbreak.;Applied and Environmental Microbiology (1985), Vol. 50, Issue 4, p.1094-1096).

Calles-Enriquez et al. (Sequencing and Transcriptional Analysis of theStreptococcus thermophiles Histamine Biosynthesis Gene Cluster: FactorsThat Affect Differential hdcA Expression; APPLIED AND ENVIRONMENTALMICROBIOLOGY, September 2010, Vol 76, No. 18, p. 6231-6238) describehistamine-producing strains of Streptococcus thermophiles, athermophilic starter used for the production of yogurt and certaincheese varieties. They further indicate that the presence of strainswith the capacity to decarboxylate histidine could result in productscontaining histamine produced during manufacture or during storagebefore consumption and that this underlies the importance of using onlyhistamine-negative strains in the manufacture of fermented dairyproducts.

Even if it has been previously known that some Lactobacillus can producehistamine it has certainly not been known that the ability to producehistamine is a key factor to assure certain health benefits for it'shost, for example anti-inflammatory properties of certain Lactobacillusstrains.

Neither is it known or obvious from the prior art that this can be usedfor screening and selection of certain probiotic strains ofLactobacillus.

Mast Cells

A mast cell (also known as mastocyte and labrocyte) is a resident cellof several types of tissues and contains many granules rich in histamineand heparin. Although best known for their role in allergy andanaphylaxis, mast cells play an important protective role as well, beingfor example intimately involved in wound healing and defense againstpathogens.

Mast cells are present in most tissues characteristically surroundingblood vessels and nerves, and are especially prominent near theboundaries between the outside world and the internal milieu, such asthe skin, mucosa of the lungs and digestive tract, as well as in themouth, conjunctiva and nose.

In allergic reactions, mast cells remain inactive until an allergenbinds to IgE already in association with the cell. Other membraneactivation events can either prime mast cells for subsequentdegranulation or can act in synergy with FceRI signal transduction.Histamine from such granulation dilates post capillary venules,activates the endothelium, and increases blood vessel permeability.Histamine release leads to local edema (swelling), warmth, redness, andthe attraction of other inflammatory cells to the site of release. Italso irritates nerve endings (leading to itching or pain). Cutaneoussigns of histamine release are the “flare and wheal”-reaction. The bumpand redness immediately following a mosquito bite are a good example ofthis reaction, which occurs seconds after challenge of the mast cell byan allergen. The other physiologic activities of mast cells are muchless well-understood. Several lines of evidence suggest that mast cellsmay have a fairly fundamental role in innate immunity—they are capableof elaborating a vast array of important cytokines and otherinflammatory mediators such as TNFα, they express multiple “patternrecognition receptors” thought to be involved in recognizing broadclasses of pathogens, and mice without mast cells seem to be much moresusceptible to a variety of infections.

Considering the toxicity of bacterial histamine in foods and the factthat it is recommended to avoid histamine-producing strains in fermentedproducts (see further examples in Calles-Enriquez et al. Sequencing andTranscriptional Analysis of the Streptococcus thermophiles HistamineBiosynthesis Gene Cluster: Factors That Affect Differential hdcAExpression; APPLIED AND ENVIRONMENTAL MICROBIOLOGY, September 2010, Vol.76, No. 18, p. 6231-6238) it can not considered to be obvious to usecertain selected Lactobacillus strains for local production of histaminein treatment and/or prophylaxis of various diseases.

The relationship between a host and its microbes is complex, as it alsois for certain of a mammalian's own cell types, such as mast cells. Thishost-microbe relationship has been developing over many years ofco-evolution; this includes the microbes production of variousmetabolites that can benefit the host nutritionally, immunologicallyetc., act as whole or part antagonists, agonist, de-sensitization etc.of specific receptors or other processes. There is therefore also a needto understand such specific interactions between microbes and manrelated to a specific disease or other situations influencing the healthof the host so that the most appropriate probiotic strains can beselected and used to counteract such developments.

The inventors herein have found that a selected group of lactobacilli,including certain strains of Lactobacillus reuteri locally produceshistamine under specific growth conditions. Such locally producedhistamine, contrary to earlier belief, will benefit the host in multipleways including reduction of inflammation, reducing certain cancers etc.

Another object of the invention is to provide products containing saidstrains together with a specific carbon source, in order to have asymbiotic product.

Other objects and advantages will be more fully apparent from thefollowing disclosure and appended claims.

Administering the lactic acid bacterial strains, selected accordingly tothe present method, to a mammal will result in locally producedhistamine that could be beneficial for several reasons.

A primary object of the present invention is to provide a method ofselecting lactic acid bacterial strains assuring good anti-inflammatoryeffect. These strains could preferably be used for treatment and/orprophylaxis of inflammatory conditions, since the histidine operon andthe production of histamine is essential for the anti-inflammatorycapacity of certain lactic acid bacterium. Preferably the strains may beused for treatment and/or prophylaxis of inflammatory processes in theGI tract, GU tract, oral cavity, in the lungs and airways, on the skinetc, of the mammalian body, including but not limited to colitis, IBD,IBS, diverticulosis, gingivitis, vaginitis etc. It is previously knownthat histamine via the H2 receptor can reduce the gene expression ofTNF-alpha. Further, mast cells are capable of elaborating a vast arrayof important cytokines and other inflammatory mediators such asTNF-alpha. However it is not previously known that the histidine operon,and local histamine production of such selected strains, could bebeneficial to the host and is for example a key factor in selected L.reuteri strains' anti-inflammatory capacity. Neither is it previouslyknown to use L. reuteri selected according to the present method intreatments requiring histamine.

Preferred products and strains for the treatment and/or prophylaxis ofinflammatory conditions are Lactobacillus reuteri, in particularLactobacillus reuteri 6475 (ATCC PTA 6475). In other embodiments of theinvention the strain used is not Lactobacillus reuteri 6475 (ATCC PTA6475).

The therapeutic uses of the strains, products and compositions of theinvention as defined herein generally result in the reduction oralleviation of the relevant disease or symptoms of disease, for examplecan result in a significant reduction in inflammation levels in themammal. For example, locally produced histamine may be activating H₂receptors on intestinal epithelial cells as well as immune cells tosuppress host mucosal immunity, e.g. via the inhibition ofpro-inflammatory cytokines. Thus, the present invention allows for theconversion of a dietary component (histidine) to histamine at the siteof activity and local modulation of the host immune response (e.g. inthe gut). It can be seen that such local production of histamineprovided by the present invention can provide real advantages over forexample oral ingestion or other forms of administration of histamine,especially given the fact that such oral ingestion would not beadvocated due to the recognized toxic effects and health risks.

In particular where inflammatory diseases of the intestine areconcerned, the therapeutic uses of the strains, products andcompositions of the invention can result in significant reduction inulceration and intestinal damage (e.g. colon damage) measured forexample by a standard method such as a Wallace score, a significantreduction in weight loss or a significant reduction in inflammation ofthe intestine, e.g. the colon.

Such reduction or alleviation of disease or symptoms thereof can bemeasured by any appropriate assay. Preferably the reduction oralleviation of disease or symptoms is statistically significant,preferably with a probability value of <0.05. Such reduction oralleviation of disease or symptoms is generally determined compared toan appropriate control individual or population, for example a healthymammal or an untreated or placebo treated mammal.

An appropriate mode of administration and formulation of the strains,etc., is chosen depending on the site where local production ofhistamine is desired. A preferred mode of administration is oral,however, equally for some treatments topical or some other form of localadministration to the skin, rectum, vagina or gums will be appropriate,or intravenous or intramuscular injection will be appropriate.

Although the Examples herein demonstrate the use of strains of theinvention and appropriate doses thereof to treat colitis, it will beappreciated that this is only one example of the inflammatory conditionswhich can be treated in accordance with the present invention and thatappropriate doses of the strains, products and compositions of theinvention as defined herein can be chosen depending on the disease to betreated, the mode of administration and the formulation concerned.

Dietary mixtures comprising histidine may be used to ensure the presenceof histidine and thereby increase the efficacy of the bacteria.Histidine may be administered alone or together with the bacteria.

One possibility to ensure the bacteria's supply of histidine is to eathistidine rich food, including but not limited to soy protein, cheese,egg, chicken and pork.

The histidine operon in bacteria has been shown to improve the growthcapacity under conditions of low pH or energy source limitation(Calles-Enriquez et al.) but the histidine operon has not beenassociated with anti-inflammatory features of certain L. reuteristrains.

It is another object of the present invention to use the strainsselected according to the present method in cancer therapy. Histamine incombination with IL-2 has been used for treatment of AML. Using thestrains of the present invention will result in locally producedhistamine that in combination with IL-2 could be used for treatment ofAML.

Another object of the present invention is to use the selected strainsin order to reduce food allergy, other allergic reactions or otherautoimmune diseases. Systemic increases in histamine are as previouslyknown a consequence of allergy by the granulation of mast cells. Whenadministering strains selected according to this invention to arecipient, the locally produced histamine will lead to a desensitizationeffect that will reduce allergy or other autoimmune diseases.

It is also an object of the present invention to use thehistamine-producing lactic acid bacteria strains to reduce the risk oftraveler's diarrhea. Patients treated with histamine blockers have anincreased risk of getting traveler's diarrhea; this increased risk couldbe neutralized by administrating the lactic acid bacteria selectedaccording to the present method.

Yet another object of the present invention is to use the selectedstrains in treatment of MS. Histamine has been proposed to be animportant molecule for developing new treatments for MS and the strainsselected accordingly to the present invention will provide the patientwith histamine.

Yet another object of the present invention is to usehistamine-producing bacteria as a skin-anti-inflammatory treatment usingavailable histidine and histidine analogs in the skin. Since histidineis a substrate for urocanic acid which the skin produces by UVirradiation and the urocanic acid has anti-inflammatory properties onthe skin.

It is another object to use such selected strains to inhibit activationof ERK1/2.

Another object of the invention is to inhibit TNF alpha.

Another object of the invention is to reduce inflammation, locally orsystemically.

Another object of the invention is to enhance synaptic vesicleexocytosis by inhibiting ERK1/2.

Another object of the invention is to promote human embryonic stem cellself-renewal by inhibiting ERK1/2.

Another object of the invention is to induce macrophage ABCA1 expressionand cholesterol efflux by inhibiting ERK1/2.

Another object of the invention is to reduce cardiac hypertrophy andheart failure by inhibiting ERK1/2.

Another object of the invention is to reduce the proliferation ofcertain cancers including leukemia (for example AML) or malignantmelanoma. Thus, such cancers are preferred diseases which could betreated using the strains, products and compositions of the presentinvention.

Another object of the invention is to use selected strains to producehistamine under certain conditions as a neurotransmitter for example inGU tract interactions with the CNS, and also neural signaling in localpain. This role as a neurotransmitter can be extended to effects onintestinal motility (to treat constipation or diarrhea) and to painsignaling in the gut.

Another object of the invention is to use selected strains forinfluencing the gut-brain axis as the selected lactic acid bacteria(LAB) will produce histamine and affect visceral pain perception andsignaling in the enteric nervous system. Thus, it can be seen that thepresent invention can be used for the treatment and/or prophylaxis ofany disease which will benefit from local histamine production or thetreatment and/or prophylaxis of any disease which can be treated withthe local administration of histamine.

It is a further object of the invention to provide products containingsaid strains.

It is a further object of the invention to provide products containingsaid strains together with a specific carbon source, in order to have asynbiotic product, which will through specific stimulation of thehistamine-producing strain, enhance the effects.

It is a further object of the invention to provide products comprisingthe said strains together with histidine, including histidine analogs orhistidine contained in products or compositions. Preferably such amixture is administered orally in a protective capsule for release ofthe content in the lower GI tract to ensure survival of both thehistidine and the bacteria at the site of action.

It is a further object of the invention to combine administration ofsaid strains with a histidine rich diet.

A yet further aspect of the invention provides a product for thetherapeutic uses as defined elsewhere herein, wherein said use furthercomprises the administration of at least one further therapeutic ornutritional agent. In such embodiments, the further therapeutic agentcan be any further agent which is useful in the treatment of the diseasein question, for example is a further anti-inflammatory agent or animmunotherapeutic agent such as for example a chemokine or cytokine(e.g. IL-2).

In preferred embodiments, said further agent comprises histidine or ahistidine analog, an appropriate carbon source which supports theproduction of histamine by the bacterial strain, or a combinationthereof.

Said further agents can be administered together with the strains of theinvention or can be administered separately. In addition, said furtheragents can be administered at the same time as the strains of theinvention or at different time points. Suitable administration regimesand timings can readily be determined by the skilled person depending onthe further agent in question.

The present invention also provides a composition comprising:

(i) a lactic acid bacterial strain obtainable by the selection method ofthe invention (or a lactic acid bacterial strain capable of producinghistamine as otherwise defined herein), wherein said lactic acidbacterial strain has an active histidine operon and is capable ofproducing histamine; and(ii) at least one additional component selected from the groupconsisting of an appropriate carbon source which supports the productionof histamine by said strain, a source of histidine or histidine analog,and a combination thereof.

In products, compositions and uses of the invention as described herein,preferably said histidine or histidine analog is in the form of ahistidine or histidine analogy contained in a foodstuff or foodsupplement, or said carbon source comprises glucose. Preferably saidcarbon source will not comprise sucrose, or at least will only comprisesucrose at such a level which will not significantly compromisehistamine production by the strain. Optionally, sources of other aminoacids can also be provided.

In alternative embodiments, the strains as defined in part (i) can becombined with a further component which is useful in the treatment ofthe disease in question, i.e. a further therapeutic agent, for example afurther anti-inflammatory agent or an immunotherapeutic agent such asfor example a chemokine or cytokine (e.g. IL-2).

Lactobacillus reuteri is a heterofermentative lactic acid bacterialspecies that naturally inhabits the gut of humans and animals. Specificprobiotic L. reuteri strains potently suppress human TNFα productionwhile other probiotic L. reuteri strains enhance human TNFα production.

The invention herein is made possible by mechanistic studies ofprobiotic L. reuteri strain 6475 and other strains which havedemonstrated their effect upon activated human myeloid cells. L. reuterimetabolites were isolated using HILIC-HPLC, and histamine was identifiedby NMR spectroscopy and mass spectrometry. Quantification of histamineby triple quadrupole MS revealed that L. reuteri strain 6475 producesrelatively high concentrations of histamine when grown in aglucose-based minimal media. Previous transcriptomics studies hadsuggested that two genes in the L. reuteri histidine operon may play arole in TNF inhibition by strain 6475. Targeted mutagenesis of thesegenes revealed that each gene in the histidine operon, thehistidine/histamine antiporter, HdcA, and HdcB, are important for theTNF-inhibitory phenotype of strain 6475. Mechanistic studiesdemonstrated that histamine is inhibiting TNF via signaling through theH₂ but not H₁ receptor. Signaling through the H₂ receptor increasesintracellular cAMP, which activates PKA. PKA activity is necessary forTNF suppression by histamine. Histamine blocks activation of the MEK-ERKMAPK signaling pathway.

Histamine is better known for its pro-inflammatory effects in allergyand anaphylaxis, but several studies have demonstrated anti-inflammatoryfunctions of histamine. In vitro studies have shown that histamine caninhibit production of pro-inflammatory cytokines, IL-1, IL-12, and TNFfrom LPS-stimulated human monocytes and macrophages and this effect isreversed by H₂ receptor antagonists. Additionally, histamine canstimulate production of the anti-inflammatory cytokine, IL-10, via theH₂ receptor. Signalling through the H₂ receptor results in decreasedexpression of the CD14 receptor, a receptor involved in LPS recognition,on the surface of human monocytes. The TNF receptor is also affected byhistamine. Signalling through the H₁ receptor induces shedding of boththe TNFR1 and the TNFR2. In vivo studies have also revealed ananti-inflammatory role for histamine. Treatment with dimaprit, aspecific H₂ receptor agonist, reduced plasma TNF levels in mouse modelsof endotoxin shock (LPS challenge) and hepatitis (LPS plus galactosaminechallenge). Histamine was protective in an LPS-induced liver injurymouse model, and these effects were attenuated in an H₂ receptorknock-out mouse. In the gut, histamine may help protect againstbacterial infection. Signalling through the H₂ receptor in Peyer'spatches helps prevent infection by Yersinia enterocolitica.

The effect of histamine can be determined by the expression of histaminereceptors on the target cell. In T-cells, the effect of histamine isdependent on which histamine receptor is activated.

By signalling through the H₁ receptor, histamine enhances T_(H)1-typeresponses but suppresses both T_(H)1 and T_(H)2 responses via the H₂receptor. A study was performed looking at histamine receptor expressionin the human gastrointestinal tract. Many of the cell types examinedexpressed multiple histamine receptors. For example, immune cells,including macrophages, highly expressed the H₁ and H₂ receptor anddemonstrated low expression of the H₄ receptor. Increased mast cells andhistamine have been implicated in the visceral hypersensitivityassociated with IBS. The increased number and activity of mast cellsnear colonic mucosal innervation may result in heightened abdominal painperception. A study with ketotifen, a mast cell stabilizing agent,demonstrated an increased pain threshold in patients with IBS, decreasedIBS symptoms, but no change in the number or activity (determined byhistamine and tryptase release) of mast cells in rectal biopsy tissue.The effects of ketotifen in improving IBS may not be the result ofstabilizing mast cells, but could be attributed to its other role as anH₁ receptor antagonist. If activation of the H₁ receptor is associatedwith a pro-inflammatory response, blocking its activity with ketotifencan allow histamine produced either by mast cells or the gut microbiota,such as L. reuteri, to signaling through the H₂ receptor only. As wehave demonstrated, signaling via the H₂ receptor can suppress TNFproduction and cause an anti-inflammatory effect. This ketotifenmechanism can be used for new therapies combining H1 receptorantagonists with general probiotic effect of a L. reuteri strain.

Further changing the carbon source of the growth media from glucose tosucrose is sufficient to suppress the TNF-inhibitory phenotype of aselected strain, for example L. reuteri strain 6475. In addition,significant down-regulation of all three genes in the histidine operonwas observed with the sucrose growth condition.

The identification of histamine as an anti-inflammatory compoundproduced by selected probiotic Lactobacillus strains will help determinetherapeutic applications for such strains. Mechanistic studies linkedthe activation of the H₂ receptor on THP-1 cells with histamine and thesuppression of ERK activation. ERK activation is involved in manycellular functions besides TNF production. ERK activation is involved inproliferation, tumorigenesis, differentiation, and cell survival. Theresults suggest a role for selected strains such as L. reuteri 6475 inprotecting against cancer by suppressing inflammation, cellproliferation, and apoptosis via inhibition of ERK activation. Inaddition, histamine is a known neurotransmitter. Production of histamineby selected strains can influence signalling in the enteric nervoussystem, impacting pain perception and gut motility. To ensure theproduction of histamine at the site of action it may be advantageouslyto provide the bacteria with histidine. Histidine may be administeredtogether with the bacteria or alone, diets rich in histidine mayincrease the histamine production as well.

The present invention provides certain strains of lactic acid bacteriaand a method of selecting such strains and products comprising suchstrains. The bacteria are selected using a screen for the histidineoperon; surprisingly the presence of an active histidine operon has beenshown to be essential for various beneficial effects such as theimmunomodulatory properties of lactic acid bacterial strains.

Other objects and advantages of the present invention will becomeobvious to the reader and it is intended that these objects andadvantages are within the scope of the present invention.

The invention will be further described with reference to the followingnon-limiting Examples:

EXAMPLES

TABLE 1 Bacterial strains used in this study Bacterial StrainsDescription Source L. reuteri ATCC PTA Isolate from Finnish BioGaia AB6475 mother's milk (Raleigh, NC) L. reuteri ATCC PTA insertional mutantThis study 6475::JP577 L. reuteri ATCC PTA insertional mutant This study6475::1229 L. reuteri ATCC PTA insertional mutant This study 6475::1230L. reuteri ATCC PTA insertional mutant This study 6475::1231

TABLE 2 Transcriptomic analysis of the histidine operon in L. reuteristrain 6475 mutants Histidine decarboxylase, Histidine/Histamine HdcBgene pyruvoyl type A (HdcA) Antiporter Comparison Fold Change p ValueFold Change p Value Fold Change p Value CFAS*(23)/6475 −2.3 <0.05 −1.10.68 −1.2 0.36 THFS1*/6475 −1.33 0.60 −3.28 <0.05 −3.45 <0.001Sucrose^(§)/Glucose −11.8 <0.01 −30.1 <0.001 −5.5 <0.001 THFS2/6475 — —1.2 0.7 0.8 0.2 PocR/6475 — — 2.4 0.10 1.3 0.66 *Insertion mutants thatlose the ability to inhibit TNF production compared to the wild-typestrain 6475. CFAS: cyclopropane fatty acid synthase, THFS1:tetrahydrofolate synthase 1, THFS2: tetrahydrofolate synthase 2.^(§)Wild-type 6475 grown in LDMIIIS compared to wild-type 6475 grown inLDMIIIG. Wild-type 6475 grown in LDMIIIS loses the ability to inhibitTNF production.

Example 1 Production of Histamine by Selected Lactobacillus BacterialStrains and Culture Conditions

All bacterial strains used in this study are described in Table 1.Lactobacillus reuteri ATCC PTA 6475 is an isolate from Finnish mother'smilk (BioGaia AB, Raleigh, N.C.). This strains is available to thepublic at the American Type Culture Collection (Rockville, Md.) havingbeen deposited there on Dec. 24, 2004. This deposit meets therequirements of the Budapest Treaty.

L. reuteri strains ATCC PTA 6475, ATCC 6475 JP577, ATCC 6475 1229, ATCC6475 1230, and ATCC 6475 1231 will be referred to as strains 6475,JP577, 1229, 1230, and 1231, respectively, throughout this disclosure.L. reuteri strains were cultured under anaerobic conditions for 16-18 hin deMan, Rogosa, Sharpe media (Difco, Franklin Lakes, N.J.), andinoculated into 2 L of a semi-defined media, LDMIII (OD₆₀₀ adjusted to0.1), which has been described previously. The carbon source was eitherglucose, LDMIIIG, or sucrose, LDMIIIS. The culture was grown for 24 h at37° C. in an anaerobic workstation (MACS MG-500, MicrobiologyInternational, Frederick, Md.) supplied with a mixture of 10% CO₂, 10%H₂, and 80% N₂. Samples were taken at different times to follow thegrowth by measuring OD₆₀₀. At stationary phase (24 h), the cells werepelleted from the 2 L culture (4000× g, 10 min). Cell pellets andbacteria cell-free supernatants were stored at −20° C. before furtherprocessing for HPLC separation and testing in a TNF inhibition bioassay.

Cell Line and Reagents

In vitro experiments were performed with THP-1 cells (human monocytoidcell line, ATCC, Manassas, Va.) maintained in RPMI (ATCC) andheat-inactivated fetal bovine serum (Invitrogen, Carlsbad, Calif.) at37° C., 5% CO₂. MEK1/2, phospho-MEK1/2, ERK1/2, and phospho-ERK1/2antibodies and MEK inhibitor U0126 were received from Cell SignalingTechnology (Danvers, Mass.), and the β-Actin antibody was received fromAbcam (Cambridge, Mass.). All other reagents were received from Sigma(St. Louis, Mo.) unless otherwise stated.

HILIC-HPLC Separation of Cell Wall Associated Factors

Cell pellets (7 g) from strain 6475 grown in either LDMIIIG or LDMIIISwere washed with 30 mL ice cold 50% acetonitrile/0.1% trifluoroaceticacid (TFA). The cell suspension was centrifuged for 10 min, 4000×g at 4°C. Supernatants were filtered through polyvinylidene fluoride (PVDF)membrane filters (0.45 μm pore size, Millipore, Bedford, Mass.),lyophilized, and resuspended in 10 mL 0.1% formic acid. The resuspendedsample was size fractionated with Amicon Ultra-15 centrifugal filterunits using ultracel-3 membrane (Millipore, Bedford, Mass.). Thefiltrate (9 mL) was dried down to 1 mL with a speed vacuum, and 0.75 mLwas used for HILIC-HPLC. The sample was dissolved with 100% acetonitrilebefore running on a PolyLC Hydroxyethyl column with a gradient of 100-0%acetonitrile, 0.1% formic acid. The sample was run for 25 min and 25fractions (A1-C1) were collected at 10 mL/min/tube. Three millilitersfrom each fraction was lyophilized, resuspended in 3 mL 0.1% aceticacid, and lyophilized again for testing in a TNF inhibition bioassay

TNF Inhibition Bioassay and TNF ELISA

Bacterial supernatants (10 mL) from a 24 h LDMIII culture werefilter-sterilized using PVDF membrane filters (0.22 μm pore size,Millipore) and size fractionated as described above. One milliliter ofthe <3 kDa filtrate was speed vacuum dried and resuspended in RPMImedia. These processed supernatant samples are termed conditioned media.All supernatants were normalized by volume to an OD₆₀₀=1.0. Lyophilizedfractions from the HILIC-HPLC separation were resuspended in 400 μL 10mg/mL ammonium bicarbonate, speed vacuum dried, and resuspended in 400μL RPMI media. Conditioned media and cell pellet wash fractions weretested for their ability to modulate TNF production in monocytoid cells.In brief, THP-1 cells (approximately 5×10⁴ cells) were stimulated toproduce TNF by the addition of 100 ng/mL Pam₃Cys-SKKKK×3 HCl (EMCMicrocollections, Tuebingen, Germany) as previously described.Inhibitors—H₂ receptor antagonists, ranitidine and cimetidine (10⁻⁴-10⁻⁶M), H₁ receptor antagonist, indomethacin (10⁻⁵-10⁻⁶ M), MEK inhibitor,U0126 (10 μM), and PKA inhibitor, H89(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride) (10⁻⁵ M)—were added to the THP-1 cells followed by L.reuteri conditioned media or cell pellet wash fractions (5% v/v),histamine (10⁻⁵ M), or dibutyryl cAMP (10⁻³-10⁻⁷ M). Plates wereincubated at 37° C. and 5% CO₂ for 3.5 h. THP-1 cells were pelleted(3000× g, 5 min, 4° C.), and quantitative ELISAs were used to determineTNF quantities in THP-1 cell supernatants according to themanufacturer's instructions (R&D Systems, Minneapolis, Minn.).

L. reuteri 6475 TNF-Inhibitory Compound(s) were Isolated UsingHydrophilic Interaction Liquid Chromatography-High Performance LiquidChromatography (HILIC-HPLC)

Bacterial cell pellets were washed to remove compounds looselyassociated with the cell surface. Components of the cell pellet washwere separated based on hydrophobicity using HILIC-HPLC, and theresulting 25 fractions were tested for retention of the TNF-inhibitorycompound. L. reuteri 6475 grown in a minimal media with glucose as thesole carbon source produces TNF-inhibitory factors that were retained in3 separate HILIC-HPLC fractions (B3, B5 and B6, data not shown). L.reuteri 6475 grown with sucrose as the sole carbon sources loses theTNF-inhibitory phenotype and served as the negative control. None of theHILIC-HPLC fractions from the 6475 sucrose cell pellet wash demonstratedsignificant TNF inhibition (data not shown).

Histamine was Identified in a TNF-Inhibitory HILIC-HPLC Fraction by NMRSpectroscopy and Mass Spectrometry

TNF-inhibitory HILIC-HPLC fraction B3 was analyzed by ¹H NMR andcompared to the neighboring non-TNF inhibitory fraction B4. A uniqueseries of peaks with a chemical shift between 7.0-7.5 ppm, which ischaracteristic of an aromatic compound, was observed in fraction B3 butnot fraction B4 (data not shown). This aromatic compound cluster wasfurther analyzed with heteronuclear single quantum coherence (HSQC)2-dimensional (2D) NMR in order to identify its components. The aromaticcompounds consisted of tryptophan, phenylalanine, histamine, and onecompound that was unidentifiable. Tryptophan and phenylalanine arecomponents of the bacterial growth media while histamine is not. Theseresults were confirmed using an additional 2D NMR method, totalcorrelation spectroscopy (TOCSY). Histamine is a biogenic amine that isproduced from histidine via the histidine decarboxylase by somefermentative bacteria including lactobacilli. Histamine was alsoidentified in fraction B3 using electrospray time-of-flight massspectrometry (ESI TOF MS). Histamine is not covalently modified based onits fragmentation pattern in MS/MS analysis. Analysis of thecorresponding B3 fraction of L. reuteri 6475 grown in a sucrose mediawith ESI TOF MS did not reveal any histamine. L. reuteri 6475 grown in aglucose media produces histamine, which is present in a TNF-inhibitoryHILIC-HPLC fraction.

Histamine was Quantified in Select HILIC-HPLC Fractions Using TripleQuadrupole Mass Spectrometry

Triple quadrupole mass spectrometry is an established, highly sensitivemethod of quantifying small molecular compounds. Histamine wasquantified in a select range of HILIC-HPLC fractions from L. reuteri6475 glucose (B2-B7) and sucrose (B2-B9) as well as the bacterialculture supernatant. High levels of histamine (>300 ng/mL) correlatedwith the ability of the HILIC-HPLC fractions to inhibit TNF (FIG. 1).Low levels of histamine were measured in most fractions examined,including those from 6475 sucrose (FIG. 1). The ability of histamine toinhibit TNF production appears to be concentration dependent.

Synthetic Histamine and Histamine Produced by L. reuteri 6475 InhibitTNF Production Via the H₂ Receptor

Histamine can significantly inhibit TNF production from TLR2-activatedhuman monocytoid cells (THP-1) (FIG. 4A). Histamine can signal throughfour different histamine receptors, however, monocytoid cells expresshigh levels of the H₁ and H₂ receptors only. Previous studies have showneffects of histamine on TNF production via the H₂ receptor. H₁ and H₂receptor-specific antagonists were used to determine which receptor wasmediating the effect of histamine on THP-1 cells. H₂ receptor-specificantagonists, ranitidine and cimetidine, could block TNF-inhibition byhistamine in a concentration dependent manner (FIG. 2A). Flow cytometryanalysis with H₂ receptor-specific antibodies revealed that THP-1 cellshighly express the H₂ receptor (data not shown). An H₁ receptor-specificantagonist, indomethacin, had no effect on TNF-inhibition by histamine(FIG. 2A). Histamine blocks TNF production from TLR2-activated THP-1cells via signaling through the H₂ receptor. L. reuteri 6475 conditionedmedia containing histamine significantly inhibits TNF compared to themedia control, and this effect is partially blocked by H₂ receptor butnot H₁ receptor antagonists (FIG. 2A). A partial block in TNFsuppression indicates that histamine present in 6475 conditioned mediais signaling via the H₂ receptor but that other TNF-inhibitory factorsthat act through alternative mechanisms may also be present in theconditioned media. The cell pellet wash containing histamine of strain6475 also suppresses TNF production (FIG. 2B). As seen with 6475conditioned media, H₂ receptor antagonists partially block the effect ofthe 6475 cell pellet wash (FIG. 2B), suggesting multiple immunomodulinsare present in the unfractionated cell pellet wash. The effects ofTNF-inhibitory fraction B3, which contains high amounts of purifiedhistamine, were completely blocked by the addition of H₂ receptorantagonists (FIG. 2B).

Example 2 Selection of Strains Producing HistamineIdentification/Selection of Histamine Producing Bacteria

Strains to be tested and possibly selected were cultured under anaerobicconditions for 16-18 h in deMan, Rogosa, Sharpe media (Difco, FranklinLakes, N.J.), and inoculated into 2 L of a semi-defined media, LDMIII(OD₆₀₀ adjusted to 0.1). The carbon source was glucose, LDMIIIG. Eachculture was grown for 24 h at 37° C. in an anaerobic workstation (MACSMG-500, Microbiology International, Frederick, Md.) supplied with amixture of 10% CO₂, 10% H₂, and 80% N₂. Samples were taken at differenttimes to follow the growth by measuring OD₆₀₀. At stationary phase (24h), the cells were sampled for analysis using real-time PCR to test forthe presence of the three genes, the histidine/histamine antiporter,HdcA, and HdcB.

For strains positive for the three genes, the levels of producedhistamine are determined by triple quadrupole mass spectrometry. Thestrains with highest production of histamine (>250 pg/ml) are selected.Histamine production can also be evaluated and quantified by ELISAs orimmunoassays.

Example 3 Demonstration of Immunomodulation

The Histidine Operon Contributes to the TNF-Inhibitory Phenotype of L.reuteri 6475

Three genes that appear to be part of an operon are involved inhistamine production by L. reuteri 6475. These genes are thehistidine/histamine antiporter, the histidine decarboxylase pyruvoyltype A (HdcA), and HdcB (FIG. 3A). Previous transcriptomics studiessuggested that the histidine/histamine antiporter gene and HdcA werepotentially important for the TNF-inhibitory phenotype of strain 6475.All 3 genes are strongly down-regulated in 6475 grown in a sucrose media(loses TNF inhibition) compared to 6475 grown in a glucose media (Table2). In addition, at least 1 gene in the operon is down-regulated in 2mutants that lose TNF-inhibition (Table 2). These mutants wereinvestigated previously, and even though the gene products didn't haveTNF-inhibitory properties, the genes appeared to be important for theanti-inflammatory phenotype of 6475. In contrast, 2 mutants that do notlose TNF inhibition demonstrated no down-regulation of any of the genesin the histidine operon (Table 2). Mutations were made in each of these3 genes by inserting a premature stop codon into the gene sequence(strains 1229, 1230 and 1231). A mutation was also made in an unrelatedgene, the rifampicin resistance gene, to serve as a negative control(strain JP577). A mutation in just one of the genes in the histidineoperon was sufficient to cause a partial loss of TNF-inhibition comparedto the wild-type strain (FIG. 3B), suggesting that each one of thesegenes is important for the TNF-inhibitory phenotype of L. reuteri 6475.A partial loss of activity suggests that other active immunomodulins arestill being produced by L. reuteri 6475.

ERK1/2 Activation is Essential for TNF Production by TLR2-StimulatedMonocytoid Cells

ERK1/2 is activated by phosphorylation from upstream MAPKK, MEK1/2, andhas been shown previously to be important for TNF production. THP-1cells were treated with a specific MEK1/2 inhibitor, U0126, for varyingamounts of time prior to stimulation with a TLR2 agonist to suppressERK1/2 activation. Treatment with U0126 for 30 min was sufficient toprevent TNF production (data not shown). ERK1/2 is activated followingTLR2 stimulation and important for stimulating TNF production in ourmodel system.

Stimulation of the H₂ Receptor Results in Increased cAMP within theCells

The H₂ receptor is a G protein linked receptor that can activateadenylate cyclase and increase intracellular cAMP. TNF can be inhibitedat the level of transcription by cAMP and cAMP analogs. THP-1 cells werestimulated with a TLR2 agonist in the presence of a medium control, 6475supernatant or histamine with or without an H₂ receptor antagonist andintracellular levels of cAMP were measured. L. reuteri 6475 supernatantcaused a small but significant increase in cAMP (data not shown).Treatment with an H₂ antagonist blocked this effect. An increase in cAMPwas also seen with histamine treatment, and the effect was blocked by anH₂ antagonist (data not shown). A synthetic analog of cAMP, dibutyrylcAMP (dcAMP), was added to TLR2-stimulated THP-1 cells and the effect onTNF production was monitored. The addition of dcAMP (10⁻⁵-10⁻³ M) wassufficient to inhibit TNF production (data not shown). Stimulation ofthe histamine H₂ receptor results in increased cAMP, which can blockdownstream TNF production in activated monocytoid cells.

Protein Kinase A (PKA) Activity is Important for TNF Inhibition by L.reuteri 6475, Histamine, and dcAMP

Increased concentration of cAMP can activate PKA and subsequentlyinhibit the downstream ERK MAPK signaling pathway. To determine if PKAactivity was important for TNF suppression by histamine produced bystrain 6475, activated THP-1 cells were treated with a specific PKAinhibitor, H89, in the presence of 6475 supernatant, fraction B3,histamine or varying concentrations of dcAMP. The addition of H89partially blocked TNF inhibition by all of these normally TNF-inhibitorycompounds (data not shown). PKA activity is important for suppression ofTNF by histamine and dcAMP.

Signaling Through the H₂ Receptor Blocks Activation of MEK1/2 and ERK1/2

Previous studies have demonstrated that PKA can inhibit Ras/Rafactivation of MEK and subsequently ERK MAPK signaling Treatment ofactivated THP-1 cells with 6475 supernatant, histamine or U0126 blocksphosphorylation of both MEK1/2 and downstream ERK1/2 compared to themedia control (data not shown). Treatment with an H₂ receptor antagonistrestores activation of both MEK1/2 and ERK1/2 (data not shown). Therewas no difference in MEK1/2 and ERK1/2 protein levels with any of thetreatment options. Histamine from strain 6475 inhibits activation of MEKand downstream ERK to result in decreased TNF production fromTLR2-stimulated myeloid cells.

These experiments thus show that stimulation of the H₂ receptor resultsin increased cAMP, activation of Protein Kinase A (PKA) and inhibitionof the MEK-ERK MAPK signaling pathway. As described above, mechanisticstudies were performed to determine the effect of histamine on MitogenActivated Protein Kinase (MAPK) signaling pathways. Inhibition of theMEK-ERK signaling pathway with a MEK-specific inhibitor is sufficient toblock TNF production. Treatment of activated THP-1 cells with strain6475 supernatant or histamine increased intracellular cAMP. The increasein cAMP was blocked by ranitidine, a specific H₂ receptor antagonist.Treatment of TLR2-stimulated THP-1 cells with a synthetic analog ofcAMP, dcAMP, is sufficient to inhibit TNF production. Inhibition of PKAactivity partially blocks TNF suppression by previously TNF-inhibitorycompounds 6475 conditioned media, fraction B3, histamine, and dcAMP.Treatment of activated THP-1 cells with 6475 conditioned media,histamine, or U0126 suppressed activation of MEK1/2, an effect that wasblocked in the presence of ranitidine. Treatment of activated THP-1cells with 6475 conditioned media, histamine, or U0126 suppressedactivation of ERK1/2, an effect that was blocked in the presence ofranitidine.

Example 4 MEK1/2 and ERK1/2 Detection by Western Blot

THP-1 cells were lysed in ice-cold lysis buffer consisting of 50 mMTris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 1% v/vNonidet P40, 0.2% v/v NaN₃, and protease and phosphatase inhibitors.Lysates were incubated on ice for 30 min, vortexed every 10 min, andcleared by centrifugation at 13,000×g for 10 min at 4° C. Proteinconcentrations were measured using the Quant-iT™ Protein Assay kit(Invitrogen) and a Qubit fluorometer according to the manufacturer'sinstructions. Equal amounts of proteins were loaded onto electrophoresisgels.

Analysis of ERK1/2 activation was performed using specificphospho-ERK1/2 antibodies. Cell extracts were loaded on a 10%SDS-polyacrylamide gel and transferred to polyvinylidene difluoridemembranes (Bio-Rad, Hercules, Calif.). Membranes were blocked overnightat 4° C. in blocking buffer (Li-Cor Biosciences, Lincoln, Nebr.). Afterseveral washes, membranes were probed with ERK1/2, phospho-ERK1/2 orβ-Actin specific antibodies diluted in blocking buffer (Li-Cor) for 1 hat room temperature. After washes, membranes were incubated with theappropriate horseradish peroxidase-conjugated secondary antibody for 1 hat room temperature, and blots were then developed using achemiluminescent detection. Analysis of MEK1/2 activation was performedas described above except primary antibody incubation was overnight at4° C.

Example 5 hdcA Mutant Yield Diminished Ability to Attenuate ColitisBacterial Strains and Culture

Mutants were generated using RecT-mediated oligonucleotiderecombineering. L. reuteri expressing RecT (strain RPRB0000) was used toconstruct mutations in rpoB (locus tag HMPREF0536_0828 (ZP_03961568))and the target genes located in the histidine decarboxylase gene clusterHMPREF0536_1229 (ZP_03961969), HMPREF0536_1230 (ZP_03961970) andHMPREF0536_1231 (ZP_03961971) to yield strains RPRB3002, RPRB3004,RPRB3005 and RPRB3006, respectively. Mutations were verified by PCR, andthe integrity was confirmed by sequence analysis.

L. reuteri ATCC PTA 6475 and histidine decarboxylase gene (hdcA) mutantwere cultured in deMan, Rogosa, Sharpe (Difco, Franklin Lakes, N.J.) at37° C. in an anaerobic workstation (MACS MG-500, MicrobiologyInternational, Frederick, Md.) supplied with a mixture of 10% CO₂, 10%H₂, and 80% N₂.

Preparation of L. reuteri Cells and Administration to Mice

A single colony of each of the L. reuteri strains was inoculated in 10ml of MRS medium and grown at 37° C. under anaerobic condition for 18-20hours. Bacteria adjusted to OD600=0.03 were inoculated into 40 ml of MRSto start the fermentation and grown at 37° C. under anaerobic conditionfor 5.5 hrs (OD600≈2.5, bacteria were in exponential phase at this timepoint). The cells were gently pelleted (2500× g, RT, 4 minutes) andresuspended in MRS at a concentration of 25×10⁹ CFU/ml. As a mediacontrol, sterile MRS medium was used. Each 8-week old female BALB/cmouse received one dose of freshly prepared wild type L. reuteri 6475 orhdcA mutant or MRS (0.2 ml each time) every day for seven days byorogastric gavage after 10 days of acclimatization. All mouseexperiments were performed according to an approved protocol (AN-4199;animal facility of Baylor College of Medicine). Mice (45 days old) werereceived from Harlan Laboratories (Houston, Tex.) and maintained underspecific pathogen-free conditions in filter-top cages (5 mice per cage)and had free access to distilled water and Harlan rodent chow 2918. Micewere divided into different groups randomly.

Induction of Acute Colitis Using Trinitrobenzene Sulfonic Acid (TNBS)Rectal Enema.

Colitis was induced six hours before the sixth gavage. Mice wereanesthetized by constant isoflurane inhalation. A 5% TNBS solution inwater (Sigma-Aldrich, USA) was diluted with equal volume of absoluteethanol and administered at dose of 100 mg/kg body weight intrarectally.Mice were kept head down in a vertical position for 2 minutes afterenema to ensure complete retention of enema in the colon. Procedurecontrol mice received 50% ethanol in PBS. Mice were weighed prior toTNBS administration and two days after TNBS administration. Then micewere sacrificed. Colonic inflammation and damage was determined byweight loss, macroscopic score and serum SAA concentration.

Macroscopic Assessment of Colitis

The colons were collected, opened longitudinally and images wererecorded with a digital camera. Colonic inflammation and damage weredetermined according to the Wallace criteria (Morris et al., 1989). Eachcolon was scored blindly. Statistics were performed using GraphPad Prismversion 5.01 (GraphPad Software, La Jolla, Calif.). Kruskal-Wallis testwas used to detect a significant difference among all groups included inthe analysis. Results were summarized as median and interquartile range.

Measurement of Serum Amyloid Protein A (SAA) as Systemic InflammationMarker

Blood samples were collected by cardiac puncture, anti-coagulated andcentrifuged for 10 minutes at 13,000 rpm to isolate plasma. Serumamyloid A (SAA) concentrations in plasma samples were measured usingELISA kits from ALPCO (Salem, N.H.) according to manufacturer'sinstructions. SAA is an acute phase protein indicative of systemicinflammation in mice that correlates with colitis severity.

Results

L. reuteri 6475 Protects Mice Against TNBS-Induced Acute Colitis

The anti-inflammatory effects of L. reuteri 6475 were tested in aTNBS-induced mouse model of acute colitis. Mice that received L. reuteri6475 by orogastric gavage every day were compared with mice thatreceived the media control. Mice challenged with PBS instead of TNBSwere also studied as colitis negative controls.

The FIGS. 4-6 represent data from two independent experiments. Colitisnegative controls that received PBS instead of TNBS intrarectally hadvery low weight loss (or even gained weight), rare colon damage and lowserum SAA concentrations. Colitis positive mice that received MRS mediaand TNBS/ETOH developed a severe colitis characterized by a large amountof weight loss, ulceration with inflammation in the colon and the majorsites of damage extending greater than 1 cm, and significantly elevatedSAA concentrations in serum. Orogastric gavage with L. reuteri 6475significantly reduced weight loss, macroscopic inflammation in the colonand serum SAA concentrations, showing that L. reuteri 6475 significantlyattenuated colitis.

hdcA Mutant Yields Diminished Ability to Attenuate Colitis

Using the same mouse model, we tested whether hdcA gene, which encodeshistidine decarboxylase was required for the anti-inflammatory effectsof L. reuteri 6475. 8-week old female BALB/c mice were randomly dividedinto three groups which received wild type L. reuteri 6475 or hdcAmutant or MRS media respectively. The FIGS. 7 and 8 represent data fromtwo independent experiments. Again, orogastric gavage with L. reuteri6475 significantly reduced weight loss and colon damage compared withmedia control group. Mice received hdcA mutant significantly increasedweight loss and macroscopic inflammation in the colon compared with micethat received wild type bacteria, showing that hdcA mutant yieldsdiminished ability to attenuate colitis.

While the invention has been described with reference to specificembodiments, it will be appreciated that numerous variations,modifications, and embodiments are possible, and accordingly, all suchvariations, modifications, and embodiments are to be regarded as beingwithin the spirit and scope of the invention.

1-20. (canceled)
 21. A method of treating and/or reducing the risk ofdeveloping an inflammatory disease or condition in a mammal, comprising:administering to a mammal in need thereof a lactic acid bacterial strainthat produces histamine, wherein the lactic acid bacterial strain is notLactobacillus reuteri 6475 (ATCC PTA 6475), thereby treating and/orreducing the risk of developing an inflammatory disease or condition inthe mammal.
 22. The method of claim 21, wherein the administration isoral and/or topical and provides local production of histamine.
 23. Themethod of claim 21, wherein the lactic acid bacterial strain is a strainof Lactobacillus reuteri.
 24. The method of claim 21, wherein theinflammatory disease or condition is in the gastrointestinal tract (GItract), the genitourinary tract (GU tract), oral cavity, lungs, and/orairways of said mammal, and/or is on the skin of said mammal.
 25. Themethod of claim 21, wherein the inflammatory disease or condition isselected from the group consisting of colitis, inflammatory boweldisease, irritable bowel syndrome, diverticulosis, gingivitis,vaginitis, and any combination thereof.
 26. The method of claim 21,further comprising administering at least one further therapeutic and/ornutritional agent to the mammal.
 27. The method of claim 26, whereinsaid further therapeutic or nutritional agent comprises histidine or ahistidine analog, an appropriate carbon source which supports theproduction of histamine by said strain, or a combination thereof. 28.The method of claim 27, wherein the histidine or histidine analog iscomprised in a foodstuff or a food supplement.
 29. The method of claim27, wherein the carbon source comprises glucose.
 30. A method oftreating and/or reducing the risk of developing an inflammatory diseaseor condition in a mammal, comprising: administering to a mammal in needthereof a lactic acid bacterial strain that produces histamine, whereinthe lactic acid bacterial strain that produces histamine is selected by:(a) culturing lactic acid bacteria in media comprising glucose underanaerobic conditions at 37° C.; (b) detecting whether histamine isproduced by the lactic acid bacteria by detecting the presence ofhistamine in a culture sample from the lactic acid bacteria; and (c)selecting a lactic acid bacterial strain for administration in whichhistamine is present in the culture sample, thereby treating or reducingthe risk of developing an inflammatory disease or condition in saidmammal.
 31. A method of treating and/or reducing the risk of developingan inflammatory disease or condition in a mammal, comprising:administering to a mammal in need thereof a lactic acid bacterial strainthat produces histamine, wherein the lactic acid bacterial strain thatproduces histamine is selected by: (a) detecting the presence of thehdcA gene, the hdcB gene, and/or the histidine/histamine antiporter genein the lactic acid bacteria; and/or (b)(i) culturing lactic acidbacteria in media comprising glucose under anaerobic conditions at 37°C.; and (ii) detecting whether histamine is produced by the lactic acidbacteria by detecting the presence of histamine in a culture sample fromthe lactic acid bacteria; (c) selecting a lactic acid bacterial strainfor administration in which the hdcA gene, the hdcB gene, and/or thehistidine/histamine antiporter gene is present, and/or in whichhistamine is detected in the culture sample of the lactic acid bacterialstrain.
 32. The method of claim 31, wherein the administration is oraland/or topical and provides local production of histamine.
 33. Themethod of claim 31, wherein the lactic acid bacterial strain is a strainof Lactobacillus reuteri.
 34. The method of claim 31, wherein the lacticacid bacterial strain is not Lactobacillus reuteri 6475 (ATCC PTA 6475).35. The method of claim 31, wherein the inflammatory disease orcondition is in the gastrointestinal tract (GI tract), the genitourinarytract (GU tract), oral cavity, lungs, and/or airways of said mammal,and/or is on the skin of said mammal.
 36. The method of claim 31,wherein the inflammatory disease or condition is selected from the groupconsisting of colitis, inflammatory bowel disease, irritable bowelsyndrome, diverticulosis, gingivitis, vaginitis, and any combinationthereof.
 37. The method of claim 31, further comprising administering atleast one further therapeutic and/or nutritional agent to the mammal.38. The method of claim 37, wherein said further therapeutic ornutritional agent comprises histidine or a histidine analog, anappropriate carbon source which supports the production of histamine bysaid strain, or a combination thereof.
 39. The method of claim 38,wherein the histidine or histidine analog is comprised in a foodstuff ora food supplement.
 40. The method of claim 38, wherein the carbon sourcecomprises glucose.